Herpes simplex virus 1 (HSV-1) causes painful, sight-threatening infections of the eyelid and cornea. HSV- 1 infects more than half of individuals in the United States, and 300,000 new cases of HSV-1-induced eye disease are diagnosed each year. No vaccine to reduce or prevent HSV-mediated corneal infection and blinding disease currently exists. The long-term objective of our research is to develop a prophylactic vaccine that limits the acquisition and spread of HSV-1-mediated ocular infections. An effective prophylactic vaccine against HSV-1 must be safe and highly immunogenic. Previous and preliminary studies indicate that 1) mutation of phosphorylation cluster III on ICP0 creates a markedly neuroattenuated virus (Phos3) that is immunogenic and effectively prevents corneal disease in mice;2) mutation of ICP8 creates a replication-defective virus that is immunogenic and represents a safe, live virus-based vaccine strategy;and 3) mutation of vhs enhances the immunogenicity and efficacy of a replication-defective virus. We therefore hypothesize that HSV-1 bearing mutations in phosphorylation cluster III of ICP0 can be further modified to optimize its safety and effectiveness in protecting against eye disease caused by HSV-1 by additional deletion of ICP8 and vhs. We will use an established mouse model of acute corneal infection to determine whether these multigenic alterations to HSV-1 result in a safe and optimally effective vaccine strain for reducing HSV-1-mediated corneal disease, and to determine the immunologic basis of protection. In Specific Aim 1, two new recombinant viruses will be generated, specifically to test whether ICP8 can be deleted from Phos3 without compromising its capacity to stimulate protective immune responses after immunization, and whether deletion of vhs increases the immunogenicity of the ICP8-Phos3 virus. These viruses will be used to immunize mice and will be compared with the parental Phos3 for their capacity to prophylactically protect mice against HSV-1 corneal infection. In Specific Aim 2, the magnitude and type of HSV-specific T cell responses will be determined in the lymph nodes and spleen after immunization with optimal and suboptimal vaccines, and in the cornea after HSV-1 challenge. Cytokine profiles in the cornea will be determined, and potential reactivity to a pathogenic epitope in gK will be monitored. The viral genes we have chosen to inactivate normally allow HSV-1 to replicate and/or evade the immune response of the host;thus, inactivating at least two of these genes is expected to maintain the effectiveness of this HSV-1 vaccine prototype while improving its safety. In-depth analyses of T cell responses to vaccination and challenge will provide insight into mechanisms underlying vaccine-mediated protection. PUBLIC HEALTH RELEVANCE: Herpes simplex virus 1 (HSV-1) causes painful infections of the eyelid and cornea that can threaten sight. HSV-1 infects more than half of individuals in the United States, and 300,000 new cases of HSV-1-induced eye disease are diagnosed each year. Our goal is to develop a vaccine that can be used as prophylaxis to prevent the debilitating consequences of primary and recurrent HSV-1 infections of the eye.